Prostate cancer is the most commonly diagnosed cancer and the second most leading cause of cancer deaths in American men (www.cancer.org). Most prostate cancer-related deaths are due to the metastatic form of the disease. The 5-year relative survival rate in patient's diagnosed with metastatic prostate cancer is just 28%, as compared to 100% in patient's diagnosed with localized prostate cancer. This clearly indicates the lack of effective treatment available for metastatic prostate cancer. MicroRNAs (miRNAs) are small (18~23 nucleotide long) non-coding RNAs that can influence gene expression by binding to the 3'-untranslated region of coding RNAs at the post-transcriptional level. Some miRNAs has been termed as oncomirs due to their role in promoting tumor growth, invasion and metastasis. One such oncomir is microRNA-21 (miR-21) whose levels are often up-regulated in a number of cancers, including prostate cancer. MiR-21 increases the survival and invasiveness of cancer cells by suppressing its target tumor suppressor genes, namely programmed cell death 4 (PDCD4) and maspin. Thus, drugs which target miR-21 for inhibition could provide novel treatment options for metastatic prostate cancer. Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic phytoalexin found in high quantities in various dietary sources, such as grapes, red wine, berries and peanuts. Various reports have demonstrated a significant role of resveratrol in the management of several old age diseases including cancer. The efficacy of resveratrol as an anti-cancer agent resides in its ability to interfere with cell proliferation and metastasis and enhancement of apoptosis. Resveratrol has been shown to act on several intracellular targets to exert these effects. However, the exact mechanism by which resveratrol mediates its beneficial cancer chemotherapeutic actions are not clear and is the focus of this study. Based on the reported data, we hypothesized that resveratrol mediates its anti-cancer action against metastatic prostate cancer by inhibiting the signaling pathway which involves miR-21 expression and function. To address this hypothesis, we show that resveratrol decreased cell viability, migration and invasiveness of androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. These effects of resveratrol were associated with the inhibition of miR-21, since over-expression of miR- 21 with pre-miR-21 oligonucleotides attenuated resveratrol's effect on these cells. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21 and knockdown of PDCD4 by short interfering (si) RNA reversed the resveratrol's effect on prostate cancer cells. PC-3M-MM2 cells also exhibits high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002, a known PI3-kinase inhibitor. MiR-21 expression in these cells appears to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibits the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Future investigation into the molecular mechanisms revealed that resveratrol suppressed prostate cancer growth by decreasing the levels of insulin-like growth factor-1 (IGF-1) and its receptor (R). Previous studies had associated elevated levels of serum IGF-1 with high risk of prostate cancer. IGF-1, after binding to its receptors, acts as a potent mitogen which stimulates cancer cell growth and proliferation mainly by activating Akt signaling pathway. Interestingly, this effect of resveratrol on IGF-1/IGF-1R was independent of its effect on miR-21. In summary, our data show that resveratrol exerts its anti-cancer effect on metastatic prostate cancer cells, at least in part, by targeting Akt/miR-21 pathway. These data highlight a potential molecular mechanism for resveratrol's anti-cancer action for the treatment of metastatic prostate cancer and suggest that inhibition of the IGF-1/Akt/miR-21 pathway is a rationale approach for the treatment prostate cancer metastasis.

Silencing phytochrome A1 gene (PHYA1) by RNA interference in upland cotton (Gossypium hirsutum L. cv. Coker 312) had generated PHYA1 RNAi lines with increased fiber length, strength and low micronair (finer fiber). In order to identify and characterize mRNAs and miRNAs that are differentially expressed in the RNAi plants, transcriptome and miRNAome analyses via high-throughput RNA sequencing were performed. Total RNA isolated from 10-DPA (days post anthesis) fibers and small RNAs isolated from 5-, 10-, and 15-DPA fibers of RNAi and Coker 312 lines were used to construct 6 RNA libraries and 18 small RNA libraries, respectively, which were sequenced using the Illumina HiSeq system. A total of 142 differentially expressed genes (DEGs) were identified in PHYA1 RNAi compared to Coker 312. GO analysis showed that these DEGs were mainly involved in metabolic pathways, binding and regulating enzymes (hydrolase, transferase, and oxidoreductase activities), and cell structures which were reported to play important roles in fiber development. Twenty-eight KEGG pathways were mapped for 142 DEGs, and the pathways related to glycolysis/gluconeogenesis and pyruvate metabolism were the most abundant, followed by cytochrome P450-involved pathways. Sixty-one conserved miRNA families and thirtyive novel miRNAs were identified in upland cotton. The targets of 6 conserved miRNAs, which expressed differentially in the RNAi line, were reported to participate in primary cell wall synthesis and phytohormone signaling pathways. The 35 novel miRNAs were identified in cotton for the first time, and their target genes were predicted. Nine novel miRNAs were identified to target cytochrome P450 TBP. Together, the results imply that miRNAs involved in fine-tune gene regulation might confer to the phenotype of the RNAi line with improved fiber quality. Besides characterizing mRNAs and miRNAs, the CpG site methylation status within coding regions of phytochrome genes in RNAi line in leaves and 10-DPA fibers was determined using bisulfite genomic sequencing. The PHYA1, PHYC and PHYE in RNAi line had higher methylation levels in leaves than those in Coker 312, but PHYB had lower methylation levels. In fibers, the methylation levels of PHYB also decreased in RNAi plants. However, the methylation of other phytochrome genes showed no significant changes.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24484号 / 医博第4926号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森信 暁雄, 教授 YOUSSEFIAN Shohab, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurodegenerative disease histopathologically characterized by the coexistence of amyloid plaques and neurofibrillary tangles, mainly consisting of amyloid β peptides hyperphosphorylated tau proteins, respectively. Multiple proteins and pathways are involved in the pathogenesis of AD, including Aβ precursor protein (APP), β-site APP-cleaving enzyme (BACE1), neprilysin, endothelin converting enzyme (ECE), repressor element-1 silencing transcription factor (REST), microtubule-associated protein tau, glycogen synthase kinase, and pro-inflammatory cytokines. However, how these proteins and pathways are dysregulated and converge in AD pathogenesis remains unclear. Genetic, epigenetic and environmental factors play important roles in disease progression. MicroRNAs (miRNAs), a group of small noncoding RNAs, are important epigenetic regulators that participate in AD development. We have identified three miRNAs capable of targeting several proteins in different AD-related pathways: miR-181-5p, miR-153-3p and miR-101-3p. We tested miR-181 activity with recombinant reporter gene- MME 3’-UTR constructs. All four miR-181-5p (miR-181a, miR-181b, miR-181c and miR-181d) sequences downregulated the reporter signal. Human differentiated neural cells were transfected with miR-181d-5p mimics. miR-181d-5p treatment significantly reduced MME mRNA levels, protein levels and enzyme activity. In addition, miR-181d-5p increased tau and phosphorylated tau levels proportionally. We further demonstrate that miR-153-3p reduced REST 3’-UTR activities, mRNA and protein levels in multiple human cell lines. Moreover, we show that miR-153-3p, by knocking down REST protein, induces apoptosis in HeLa cells but not differentiated neural cells. In addition, miR-153-3p regulates neuronal differentiation in neuronal stem cells, potentially via REST knockdown. We further found that miR-153 levels were correlated with a reduced likelihood of developing AD. Last, we demonstrated that miR-101-3p reduced ECE1 and GSK3β protein levels in multiple cell lines. miR-101-3p increased REST and pro-inflammatory cytokine secretion in microglia cells. In sum, we tested the hypothesis that miRNAs can serve as the master regulator of AD pathogenesis. / 2024-07-01

Chronic lung diseases including Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF) and lung cancer are major causes of morbidity and mortality in the United States due to high incidence and limited therapeutic options. In order to address this critical issue, I have leveraged RNA sequencing and integrative genomics to define disease-associated transcriptomic changes which could be potentially targeted to lead to new therapeutics. We sequenced the lung transcriptome of subjects with IPF (n=19), emphysema (n=19, a subtype of COPD), or neither (n=20). The expression levels of 1770 genes differed between IPF and control lung, and 220 genes differed between emphysema and control lung (p<0.001). Upregulated genes in both emphysema and IPF were enriched for the p53/hypoxia pathway. These results were validated by immunohistochemistry of select p53/hypoxia proteins and by GSEA analysis of independent expression microarray experiments. To identify regulatory events, I constructed an integrative miRNA target prediction and anticorrelation miRNA-mRNA network, which highlighted several miRNA whose expression levels were the opposite of genes differentially expressed in both IPF and emphysema. MiR-96 was a highly connected hub in this network and was subsequently overexpressed in cell lines to validate several potential regulatory connections. Building upon these successful experiments, I next sought to define gene expression changes and the miRNA-mRNA regulatory network in never smoker lung cancer. Large and small RNA was sequenced from matched lung adenocarcinoma tumor and adjacent normal lung tissue obtained from 22 subjects (8 never, 14 current and former smokers). I identified 120 genes whose expression was modified uniquely in never smoker lung tumors. Using a repository of gene-expression profiles associated with small bioactive molecules, several compounds which counter the never smoker tumor signature were identified in silico. Leveraging differential expression information, I again constructed an mRNA-miRNA regulatory network, and subsequently identified a potential never smoker oncomir has-mir-424 and its transcription factor target FOXP2. In this thesis, I have identified genes, pathways and the miRNA-mRNA regulatory network that is altered in COPD, IPF, and lung adenocarcinoma among never smokers. My findings may ultimately lead to improved treatment options by identifying targetable pathways, regulators, and therapeutic drug candidates. / 2017-02-01T00:00:00Z

MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs. Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations. Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages. Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases. / Ph. D.

miRNAs are important post-transcriptional regulators of gene expression which play vital roles in the arrays of physiological processes, including skin and hair follicle (HF) development. In this study, the role for miR-214 in the skin and HF development and their postnatal physiological regeneration was investigated. miR-214 exhibits discrete expression patterns in the epidermis and HF in developing and postnatal skin, and is highly expressed in the epithelial stem cells and their lineage-committed progenies. The effects of miR-214 on HF morphogenesis and cycle progression were evaluated by using doxycyclineinducible miR-214 transgenic mice (K14-rtTA/TRE-miR-214). Keratinocyte specific miR-214 overexpression during skin embryogenesis resulted in the partial inhibition of HF induction and formation of the HF reduced in size producing thinner hair. Overexpression of miR-214 in telogen skin caused retardation of the anagen progression and HF growth. Inhibitory effects of miR- 214 on HF development and cycling were associated with supressed activity of stem cells, reduced proliferation in the hair matrix, and altered differentiation. miR-214 induced complex changes in gene expression programs in keratinocytes, including inhibition of cyclins and cyclin-dependent kinases and several essential components of Wnt, Edar, Shh and Bmp signalling pathways, whereas 􀁅-catenin acts as a novel conserved miR-214 target. Indeed, the inhibitory effects of miR-214 on HF development were rescued by intracutaneous delivery of pharmacological Wnt activator. Thus, this study demonstrated that by targeting 􀁅-catenin and, therefore, interfering with Wnt signalling activity miR-214 may act as one of the upstream effectors of the signalling cascades which govern HF morphogenesis and cycling.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24912号 / 農博第2575号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 准教授 小野 肇 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

MiRNAs control skin homeostasis through post-transcriptional gene repression by binding to their target mRNAs. However, their role in regulation of apoptosis and hair loss in alopecia areata (AA) is largely unknown, which became the aim of this study. In AA mouse model (C3H/HeJ), global miRNA profiling revealed 22 miRNAs with significant changes in their expression in AA affected skin. Amongst these miRNAs, miR-486-5p was dramatically decreased in alopecic skin in both humans and mice, in striking contrast to its prominent expression in the hair follicle (HF) epithelium of healthy anagen skin. Moreover, the expression of both pri-miR-486 and miR-486 is down-regulated in the human anagen HFs and keratinocytes treated with IFN-g, one of the key factors contributing to the immune privilege (IP) collapse in HFs. Intradermal delivery of miR-486-5p mimic into mouse skin affected by AA prevented premature entrance of HFs into catagen phase and reduced the numbers of CD4+ and CD8+ lymphocytes in the peri- and intra-follicular skin compartments. Consistently, subcutaneous administration of miR-486-5p inhibitor delayed anagen progression associated with a higher number of intrafollicular NKG2D+ cells in C3H/HeJ mice. Silencing of miR-486-5p in human anagen HFs ex vivo caused premature catagen development and led to suppression of IP by up-regulating HLA class 1, IRF1, ICAM1 and CADM1 expression of which CADM1 was confirmed to be a direct target of miR-486- 5p. Transcriptome profiling of primary human epidermal keratinocytes overexpressing miR-486-5p revealed damping the signalling pathways associated with inflammatory chemokines, cytokines and interleukins. Taken together, these data suggest that miR-486-5p plays a protective role in the pathogenesis of AA by maintaining anagen phase and preventing the IP collapse. / National Alopecia Areata Foundation